An ideal clinical assay for detecting circulating tumor cells should be high throughput (as simple as a one-step reaction) and highly sensitive (requiring as little as one drop of blood). The current assay to detect circulating tumor cells is time- and labor-consuming multi-step procedure, requiring isolation of tumor cells, staining with antibodies, and repeated washes to reduce background. To overcome these obstacles, we have developed a unique aptamer probe that carries a tumor cell-activatable reporting system. The aptamer probes specifically bind tumor biomarker(s) and are optically silent in the absence of target cells, thus giving no to minimal background. To translate these research findings to the clinic, the goal of this study is to develop a novel technology for circulating tumor cell detectio in a One-Drop (of blood)-One-Step Assay (ODOSA). The specific aims of this proposal are to: 1. Optimize sensitivity and specificity of the aptamer probes for the ODOSA technology; 2. Develop a multi-sample and high-throughput platform for the ODOSA technology; and 3. Validate the ODOSA technology in clinical specimens. Scientifically, this study presents an innovative concept that tumor cells can be specifically detected by activating the aptamer probes through a natural cellular process with exclusively intracellular signals and no background. Technically, the ODOSA is a technological breakthrough over the current antibody-mediated assays. Clinically, the ODOSA provides a high- throughput and multi-sample platform, and enables physicians to detect one single circulating tumor cell among millions of cells in real time using a minimal amount of blood. There is no alternative clinical assay available to date.